System with extended range of molecular sensing through integrated multi-modal data acquisition

ABSTRACT

A multi-modal data acquisition system for detecting target material on a biological reaction surface, the system comprising a radiation source for generating an incoming beam that impinges on the biological reaction surface at an oblique incidence angle and produces a reflected beam, an interferometric detector for detecting an interferometric signal from the illuminated surface, the reflected beam being directed to the interferometric detector, a fluorescence detector for detecting a fluorescence signal from the illuminated surface; the fluorescence detector being positioned to substantially minimize the incidence of the reflected beam; and a processing system for receiving the interferometric and fluorescence signals and determining the presence or absence of target material on the biological reaction surface. A reaction surface conditioned for the simultaneous collection of fluorescence, interferometric and other signals. A multi-modal data acquisition system for collecting and processing additional modes, including multiple interferometric, fluorescence and scattering channels.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Ser.No. 60/885,698, filed on Jan. 19, 2007, entitled “Four Channel OpticalDetection on Protein-Patterned Biological Compact Disk” and to U.S.Provisional Application Ser. No. 60/916,177, filed on May 4, 2007,entitled “System with Extended Range of Molecular Sensing ThroughIntegrated Multi-Modal Data Acquisition” the disclosures of which areboth incorporated herein by this reference.

This application is related to U.S. application Ser. No. 11/675,359,filed on Feb. 15, 2007, entitled “In-Line Quadrature and Anti-ReflectionEnhanced Phase Quadrature Interferometric Detection”; U.S. patentapplication Ser. No. 10/726,772, entitled “Adaptive InterferometricMulti-Analyte High-Speed Biosensor,” filed Dec. 3, 2003 (U.S. Pat. Pub.No. 2004/0166593); U.S. Pat. No. 6,685,885, entitled “Bio-OpticalCompact Disk System,” filed Dec. 17, 2001 and issued Feb. 3, 2004; U.S.patent application Ser. No. 11/345,462 entitled “Method and Apparatusfor Phase Contrast Quadrature Interferometric Detection of anImmunoassay,” filed Feb. 1, 2006 (U.S. Pat. Pub. No. 2007/0003436); U.S.patent application Ser. No. 11/345,477 entitled “Multiplexed BiologicalAnalyzer Planar Array Apparatus and Methods,” filed Feb. 1, 2006 (U.S.Pat. Pub. No. 2007/0003925); U.S. patent application Ser. No.11/345,564, entitled “Laser Scanning Interferometric Surface Metrology,”filed Feb. 1, 2006 (U.S. Pat. Pub. No. 2006/0256350); U.S. patentapplication Ser. No. 11/345,566, entitled “Differentially EncodedBiological Analyzer Planar Array Apparatus and Methods,” filed Feb. 1,2006 (U.S. Pat. Pub. No. 2007/0023643), the disclosures of which are allincorporated herein by this reference.

BACKGROUND AND SUMMARY OF THE INVENTION

The present invention generally relates to an apparatus capable ofsimultaneous acquisition of data from multiple molecular sensingmodalities, for example and not by way of limitation, a labeled process(such as a fluorescence process) and a label-free process (such as aninterferometric process).

Generally, labeled and label-free detection modalities for molecularsensing possess complimentary advantages and drawbacks. For instance,label based sensing systems are not susceptible to background effects asmuch as label-free systems. Susceptibility to background effects oftenlimits the sensitivity of label-free systems. On the other hand, labeledsystems such as fluorescence, which is the most common moleculardetection modality in use today, suffer from low photon fluxes whichlimit their sensitivity, while label-free sensing systems (e.g., theQuadraspec biological compact disc system, such as described in the U.S.Pat. No. 6,685,885) have photon fluxes that are several orders ofmagnitude higher.

Fluorescence label based systems suffer from additional problems such asphotobleaching, which limits the ability to perform time-resolvedstudies beyond a certain length of time. Label-free systems generally donot suffer from such problems.

Label based systems require an additional chemical processing step ofattaching the “label” molecule to the molecule of interest. Thisprocess, in addition to increased processing time and cost, can alterthe behavior of the molecules of interest. Label-free systems do notrequire this additional processing step.

In spite of drawbacks such as the ones described above, fluorescentlabel based detection remains a widely used technology for molecularsensing applications, such as immunosensing and drug discovery, andpossesses high sensitivity, especially in the detection of low molecularweight analytes and even single molecule detection.

There are two main reasons for the observed performance of fluorescentlabel based systems compared to current label-free technologies. First,as mentioned earlier, fluorescent label based systems are not assusceptible to variations in background effects as label-free systems.Susceptibility to background effects can limit the sensitivity oflabel-free systems. Second, signal transduction in label-free systems isbased on some physical property of the molecule of interest, which isoften related to its molecular size. Coupled with the backgroundproblem, this molecular size dependency restricts the range of molecularsize that can be detected reliably with label-free systems. For example,detection of low-molecular weights in immunoassay continues to be achallenge for many label-free systems and they try to get around themolecular size dependency through alternate assay formats such asreverse phase or inhibition assays. While the success of such approachesin circumventing the molecular weight dependency has been demonstrated,these approaches may not always be feasible. Label based systems on theother hand rely only on the properties of the “label” molecule andconsequently work independent of the size of the molecule of interest.Thus they work equally well for large as well as small molecules andmeet the demand for low molecular weight detection in many applicationareas.

Even though fluorescent based systems have good performance compared tocurrent label-free systems, the increasing demand for multiplexing isexpected to put a significant strain on fluorescent based systems. Thisis because each molecule of interest requires a unique label. Althoughsome approaches, such as Quantum Dots, have been proposed to address the“unique label” problem, considerable understanding of their interactionwith bio-molecules will need to be built for them to emerge as aubiquitous molecular sensing format. Label-free systems do not sufferfrom this limitation and as a result are attractive from themultiplexing point of view.

From the items described above, it can be seen that the labeled andlabel-free molecular detection modalities can provide complimentaryperformance attributes. However, commercially available molecularsensing platforms do not exploit these complementary properties.Integrating these complementary molecular sensing modalities in a singleplatform can enhance the capabilities of either mode by providingcapability to perform low molecular weight detection with highsensitivity as well as the ability of multiplexing without labellimitations for suitable applications.

With this objective in mind, exemplary embodiments of systemsincorporating complementary molecular sensing modalities in a singleplatform are disclosed below. One embodiment integrates fluorescencebased detection (most widely used label based detection) andinterferometric based detection (most inherently sensitive label-freetechnology) into a single instrument. This instrument is capable ofsimultaneous data acquisition from both channels. The acquired data fromboth channels can be analyzed, and biologically relevant information,such as the amount of bound protein, can be extracted.

BRIEF DESCRIPTION OF THE FIGURES

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 shows a schematic of an embodiment of a data acquisition system;

FIG. 2 is a flowchart for an instrument control program that can be usedwith the embodiment of FIG. 1;

FIG. 3A shows an example of the results of the simultaneous acquisitionof fluorescence and interferometric data;

FIG. 3B shows an example of the correlation between fluorescence andinterferometric data from fluorescently labeled proteins immobilized onthe surface of the biological compact disc;

FIG. 4 illustrates an exemplary embodiment of an integrated fluorescenceand interferometric microarray detection system;

FIG. 5 shows specifications for a C5460-01 avalanche photodiode;

FIGS. 6A and 6B shows the spectral response and sampling frequencyresponse for the −01 avalanche photodiode;

FIG. 7 illustrates the noise character in fluorescence systems fordifferent frequencies;

FIG. 8A shows the relationship between the fluorescence excitationefficiency and the reflection coefficient of the biological compactdisc;

FIG. 8B shows the relationship between the sensitivity of in-lineinterferometric channel and the reflection coefficient of the biologicalcompact disc;

FIG. 8C shows the relationship between the sensitivity of phase contrastinterferometric channel and the reflection coefficient of the biologicalcompact disc;

FIG. 9A shows an image of the fluorescence for a biological compact diskimaged simultaneously with both fluorescence and interferometric methodson the same protein grating pattern region shown in FIG. 9B;

FIG. 9B shows an image of the in-line interferometry for a biologicalcompact disk imaged simultaneously with both fluorescence andinterferometric methods on the same protein grating pattern region shownin FIG. 9A;

FIG. 10A shows the power spectrum corresponding to FIG. 10A;

FIG. 10B shows the power spectrum corresponding to FIG. 10B;

FIG. 11A shows the imaging from the interferometric channel at differentphases in the experimental procedure in the upper four rows, and showsthe imaging from the fluorescence channel in the bottom row;

FIG. 11B shows the response curve for the analyte concentration ladderon both the fluorescence and interferometric channels.

FIG. 12A illustrates protein molecules on the biological compact diskbeing illuminated with a focused Gaussian beam, polarization beingindicated by the arrow;

FIG. 12B illustrates an angular coordinate system that can be used tocalculate the angular distribution of intensity;

FIG. 13A illustrates that the reflection change is proportional to thethickness of the protein layer when the protein layer on the surface isthin enough (much less than the probe light wavelength);

FIG. 13B illustrates that if protein molecules agglomerate on thesurface, then Mie scattering dominates and the scattering can bedetectable in the Mie scattering channel;

FIG. 14 illustrates schematically an embodiment of a four-channelmicroarray detection system that is capable of simultaneously acquiringfour different signals from protein molecules on a biological compactdisk, including fluorescence and Mie scattering channels (detected by ahigh-amplification APD), and amplitude and phase-contrast channels(interferometric channels, detected by a quadrant photodiode);

FIG. 15 shows biological compact disk images with fluorescence andinterferometric methods, the images were simultaneously captured with 4channels: (A) Fluorescence; (B) Mie scattering; (C) Amplitude and (D)Phase contrast, on the same region of a protein grating pattern with athickness of about 1˜4 nm (approx. a monolayer) that was illuminated at488 nm;

FIG. 16 shows the power spectra for the images shown in FIG. 15: (A)Fluorescence; (B) Mie scattering; (C) Amplitude and (D) Phase contrastchannels;

FIG. 17A shows the spot intensities from the interferometry andfluorescence channels;

FIG. 17B shows the response curves for both the forward and reverseassays;

FIG. 18A shows the fluorescence channel for a two-channel acquisition ofbackfilled protein stripes at a concentration of 10 ug/ml collectedsimultaneously with the interferometry channel shown in FIG. 18B;

FIG. 18B shows the interferometry channel for a two-channel acquisitionof backfilled protein stripes at a concentration of 10 ug/ml collectedsimultaneously with the fluorescence channel shown in FIG. 18A;

FIG. 18C shows the associated power spectra for the interferometry andfluorescence channel responses shown in FIGS. 18A and 18B;

FIG. 18D shows the correlation between the interferometry andfluorescence channel responses shown in FIGS. 18A and 18B;

FIG. 19A shows the fluorescence channel for a two-channel acquisition ofbackfilled protein stripes at a concentration of 10 ng/ml collectedsimultaneously with the interferometry channel shown in FIG. 19B;

FIG. 19B shows the interferometry channel for a two-channel acquisitionof backfilled protein stripes at a concentration of 10 ng/ml collectedsimultaneously with the fluorescence channel shown in FIG. 19A;

FIG. 19C shows the associated power spectra for the interferometry andfluorescence channel responses shown in FIGS. 19A and 19B;

FIG. 19D shows the correlation between the interferometry andfluorescence channel responses shown in FIGS. 19A and 19B;

FIG. 20A shows the effects of bleaching on the fluorescence channel overtime with the signal collected simultaneously with the interferometrychannel shown in FIG. 20B;

FIG. 20B shows the lack of bleaching effects on the interferometrychannel over time with the signal collected simultaneously with thefluorescence channel shown in FIG. 20A;

FIG. 20C is a graph of the average fluorescence and interferometryresponses over time corresponding to the images shown in FIGS. 20A and20B;

FIG. 21 a shows a portion of 6,800 spots printed on a region of abiological compact disc of which 3,400 are antibody spots and 3,400 arecontrol spots, each antibody spot being adjacent to a control spot;

FIGS. 21 b 1-b 3 shows scans at different times in the experimentalprocess by the interferometry channel in a simultaneous two-channel scanof interferometry and fluorescence channels: FIG. 21 b 1 shows a scan ofinitial thickness, FIG. 21 b 2 shows a scan after antigen binding; andFIG. 21 b 3 shows a scan after secondary antibody binding;

FIGS. 21 c 1-c 3 shows scans at different times in the experimentalprocess by the fluorescence channel in a simultaneous two-channel scanof interferometry and fluorescence channels: FIG. 21 c 1 shows a scan ofinitial thickness, FIG. 21 c 2 shows a scan after antigen binding; andFIG. 21 c 3 shows a scan after secondary antibody binding;

FIG. 22A shows distributions of the height increment of antibody andcontrol spots after antigen binding;

FIG. 22B shows distributions of the height increment of the antibody andcontrol spots compared with the fluorescence signal of the antibodyspots after secondary antibody binding; and

FIG. 23 shows a scaling analysis of the fluorescence and interferometrychannels.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

The embodiments of the present invention described below are notintended to be exhaustive or to limit the invention to the precise formsdisclosed in the following detailed description. Rather, the embodimentsare chosen and described so that others skilled in the art mayappreciate and understand the principles and practices of the presentinvention.

Fluorescence and interferometric signals have different angulardistributions. Fluorescence excitation leads to isotropic (but nothomogeneous) incoherent emission of radiation while the interferometricsignal comes from coherently scattered radiation in the direction of thereflected light. This difference in angular distribution can beexploited in separating signals from the two channels in the instrument.

Typically, fluorescence wavelength is longer than that of excited lightdue to energy loss of the excited molecules. The wavelength difference,called the Stokes's shift, provides another way to separate fluorescencefrom background light by using optical filters.

An exemplary embodiment of a data collection system that collects bothfluorescence and interferometric signals is shown schematically inFIG. 1. The system comprises a laser 10, a linear stage (not shown), aspin motor (not shown) (e.g., Scanner motor from Laser Lines Ltd), abiological compact disc 12, a photodetector 14, an avalanche photodiodedetector (“APD”) 16, an analog-to-digital converter (“ADC”) 18, acomputer 19 and optical components such as mirrors, filters and lens.

The biological compact-disc 12 is mounted on the spin motor capable ofspinning at user defined speeds from 20 Hz to 100 Hz in increments of 20Hz. The optical assembly is fixed to the linear stage which is capableof scanning with a resolution of 0.1 um. The combination of the spinningdisc 12 and stage translation creates a polar coordinate system forreferencing any point on the disc. Data from a given position of thelinear stage constitutes a “track” on the disc 12. Several such “tracks”are acquired with a user defined resolution, typically 20 microns, tobuild up the disc surface data in both acquisition channels.

In the embodiment illustrated in FIG. 1, the illumination laser lightfrom the laser 10 has a wavelength of 532 nm and serves as the probinglight. The incoming laser beam is focused on to the disc using a lensL3, at oblique incidence. The reflected beam containing theinterferometric signal is focused on to the photodetector 14 usinganother lens L4. Filters can be placed in front of the photodetector 14to eliminate background radiation at wavelengths different from theincident laser wavelength. As mentioned earlier, fluorescence emissionis isotropic. A fraction of fluorescence is collected using a high NAlens L1 and is focused onto the APD 16 by a lens L2. Although, inprinciple, the fluorescence emission can be separated from the probebeam and direct reflection using differences in the angular distributionof fluorescence and interferometric signals, in practice, it ispreferable to incorporate filters to eliminate stray scatter, such asfrom dust or sharp features on the disc, from making its way to the APD16.

The signals from the APD 16 and the photodetector 14 are sent to a 2channel ADC 18 for analog-to-digital conversion, after which they aresent to a computer 19. Computerized data acquisition can be done using asystem management program. A flowchart for an exemplary systemmanagement is shown in FIG. 2.

At step 20, the system devices and program are initialized for a datacollection. At step 22, the necessary scanning and data collectionparameters are obtained and stored for use in the collection process. Atstep 24, the system is moved to the starting position for datacollection. At step 26, the fluorescence signals from the APD 16 and theinterferometric signals from the photodetector 14 are sent to the ADC 18for analog-to-digital conversion, and, at step 29, the converted signalsare sent to the computer 19. At step 30, the stage is used to move theoptical assembly to the next step for further data collection. At step32, the system determines whether the current position of the stage isless than or equal to the end position for data collection. If thecurrent position is less than or equal to the end position, then controlis transferred to step 34 which starts data collection at the nextposition and transfers control to step 26. If the current position isgreater than the end position, then control is transferred to step 36which ends the collection program and shuts down the reader.

The oblique incidence design incorporated in the instrument exploits thedifferences in the angular distribution of fluorescence andinterferometric signals. The oblique design enables the spinningbiological compact disc system to possess sufficient photon fluxeswithout using multiple filters for separation of the fluorescenceemission from background light.

Many, commercial fluorescence readers use Photo-Multiplier Tubes (PMTs)for detecting fluorescence emission. PMT's necessitate tightrequirements on background shielding, protection from shock and so on.Alternatively, high-gain APDs such as the Hamamatsu C5460-01 can givecomparable performance to PMTs but without the problems mentionedearlier. One important design consideration is the bandwidth of the APDwhich limits the acquisition speed. The high-gain APD 16 used in theembodiment of FIG. 1 has a bandwidth of 100 kHz, which is sufficient toacquire data from a biological compact disc 12 spinning at 4800 rpm.

Sufficient fluorescence emission efficiency is possible over a widerange of substrates. Therefore, the biological compact disc 12 isdesigned primarily based on the requirements for the label-free systems,which in this case is interferometric. Exemplary biological compactdiscs of the present embodiment include silicon discs coated with 100 nmof silica film. These discs are useful for commercial applications,particularly as they are inexpensive to manufacture yet still exhibitrobustness and good sensitivity for all channels. Such discs areparticularly useful for in-line interferometric channel sensitivityapplications.

For the embodiment illustrated in FIG. 1, FIG. 3A shows an example ofthe readings from the simultaneous acquisition of fluorescence andinterferometric data from an exemplary biological compact disc; and FIG.3B shows an example of the quantitative correlation between thefluorescence and interferometric data. Such correlations can providevaluable information about the various biochemical processes involved indisc processing and molecular detection processes. For instance, in thisexample, fluorescently labeled antibodies are immobilized on thebiological compact disc. The fluorescence channel quantifies the amountof the antibody while the interferometric channel quantifies the sumtotal of the interactions of all molecules present in the incubatingsolution, such as buffer, salts and other chemicals. In this case, thedegree of correlation, or lack of correlation, between the two channelscan tell us about the relative strength of the parasitic process such asbinding of foreign molecules on the disc surface. Such information canhelp in optimizing disc processing steps by eliminating possibleparasitic effects. This is a unique capability made possible by theintegration of these complementary molecular detection modalities in asingle platform.

Another exemplary embodiment of a system for simultaneously acquiringfluorescence and interferometric signals on a substrate, such as aspinning microarray disk is shown in FIG. 4. Although fluorescence andinterferometric signals both have scattering effects on probing lightbeams, their scattering properties have fundamental differences, such astheir scattering distributions. With respect to interferometric signals,the scattering is mostly coherent (i.e., each molecule's scatteringlight is coherent with that of other molecules, and the total scatteringlight is coherent with incident and reflected light) and specular, inwhich Rayleigh scattering dominates. When illuminated with a coherentprobe light, scattered light from the molecules superposes in the farfield. The superposition induces the strongest intensity distribution ofthe scattering light along the reflected (specular) direction in the farfield, and the scattered light interferes with the reflected light. Assuch, the signal of the target molecules is modulated in the reflectedlight.

Fluorescence can be treated as incoherent scattered light within thedipole approximation. The incoherence is induced by the randomrelaxation time and phase of the fluorophore's excited energy level. Assuch, the fluorescence is incoherent, which means that without coherentsuperposition, the emitted fluorescence does not form a strongdirectional distribution, but rather emits into all solid angles. Itshould be noted that the distribution is also not homogenous and itsdistribution function can be determined without difficulty. Moreover,the unique spatial property of the fluorescence can help one to separatefluorescence and interferometric signals.

FIG. 4 illustrates an exemplary embodiment of an integrated fluorescenceand interferometric microarray detection system. The embodiment shown inFIG. 4 comprises a laser 40 (e.g., INNOVA300 laser from Coherent Inc.),a linear stage 42 (e.g., MM2K stage from Newport), a spin motor 44(e.g., Scanner motor from Laser Lines Ltd), a biological compact disk46, a quadrant detector 48 (e.g., PC50-6 from Pacific Silicon SensorInc.), an avalanche photodiode detector (“APD”) 50 (e.g., C5460-01 fromHamamatsu Company), an oscilloscope 52, a computer 54 and some opticalcomponents such as mirrors, filters and lens. This embodiment isdesigned to acquire three channels simultaneously. The quadrant detector48 is responsible for two interferometric channels, and the APD detector50 acquires fluorescence signals from an analyte on the biologicalcompact disk 46.

For mapping a whole disk, two free coordinates are established to form apolar coordinate system. The spinning motor 44 is used to rotate thebiological compact disk 46 and serves as the angular coordinate when themotor spins in a selectable frequency ranging from about 20 Hz to about80 Hz. The linear stage 42 serves as the polar coordinate and moves backand forth with 0.1 μm linear precision and 300 mm maximum traveldistance. The motor 44 is fixed to the linear stage 42 so thattwo-dimensional mapping can be realized with appropriate computercontrol. This system is capable of mapping a 100 mm diameter biologicalcompact disk in about 30 minutes with 2 μm by 2 μm pixel resolution.

In this embodiment, the illumination from the laser 40 has a wavelengthof about 488 nm and serves as the probing light. The laser beam isfiltered, steered and focused onto the surface of the biological compactdisk 46 with a filter, several mirrors and one 10 cm convex lens. Theradius of the focal spot is about 20 μm on the disk 46; however, higherresolution can be achieved by switching to a 10 cm short focal lengthlens or even a microscope objective lens. The reflected light is guidedinto the quadrant detector 48, which is responsible for acquiring theinterferometric signals (i.e., phase contrast and in-line signals), anda 4 cm convex lens above the disk 46 gathers fluorescence and sends itinto an APD 50 equipped with a 510 nm long-pass optical filter to blockthe scattered laser light.

An oscilloscope 52 is responsible for acquiring the waveform for eachscanned track of the disk 46. The APD 50 and quadrant detector 48 areconnected with three channels (e.g., channels 1, 2, 3) of theoscilloscope 52 via coaxial cables (see the lines from the oscilloscopeto the detectors in FIG. 1). Two of these cables are for the quadrantdetector 48 so that it can acquire the two types of interferometricsignals (i.e., phase contrast and in-line signals). Another coaxialcable connects the spin motor 44 to the oscilloscope 52. The spin motor44 generates a trigger signal for the oscilloscope 52. The computer 54controls the movement of the linear stage 42 and records data from theoscilloscope 52. Two SCSI cables (see the lines from the computer inFIG. 1) are used to connect the computer 54 to the linear stage 42 andto the oscilloscope 52.

The embodiment shown in FIG. 4 has an oblique incidence design tobenefit from the two distinct solid angular emission distributionscaused by the different coherent properties of the fluorescence andinterferometric signals. In an oblique incidence design, the probe laserbeam is adapted to be incident obliquely and focus on the surface of thebiological compact disk 46 to collect fluorescence with the convex lensabove the disk. In this configuration, the reflected light does notenter the fluorescence collection lens, but the fluorescence can beacquired with good efficiency. Meanwhile, interferometric signals can bedetected via acquiring the reflected probing light. In this way, twotypes of signals are detected simultaneously without influencing eachother.

As opposed to an oblique incidence design, traditional fluorescencedetection systems collect fluorescence and reflected probing lighttogether and then separate them with optical filters. While this methodis practical for most common biosamples, when applied to a biologicalcompact disk, the detection efficiency of the fluorescence can be lowfrom the fluorophore monolayer (1˜10 nm thickness) on the surface.Empirically speaking, the photon flux ratio between the fluorescence andthe probe light is about 1:10⁷. If the reflected probe light is mixedinto the fluorescence channel, the extremely strong background lightcauses a large influence on the fluorescence detection precision.However, it has been found that if one or more long-pass filters areused, the background light can be minimized. In order to decrease thebackground light to a reasonable extent, 4 to 5 filters (as a filterstack) may be used in some embodiments.

Since the photon flux of the fluorescence is low (about 1 nw forcollection efficiency), the addition of the filter stack can negativelyinfluence the low flux. More particularly, fluorescence flux isdecreased by 25% for each filter used (i.e., when four filters are used,only 0.4% fluorescence survives). Moreover, the probe laser is not 100%pure. For instance, the INNOVA300 Argon laser generates mostly 488 nmwavelength light with 0.1 W operation power. However, there are stillsome long light wavelengths (e.g., 514 nm and 528 nm) with a ratioconstituent above 0.01%. These wavelengths are inside the spectrum bandof fluorescence so they are mostly immune to the 510 nm LP filter. Assuch, extra optical filters would be needed to purify the laser beambeforehand.

Oblique incidence greatly minimizes the above-mentioned issues. Moreparticularly, since reflected probe light does not affect fluorescence,only one filter is needed for wavelength filtering, and only one, orpossibly even zero, optical filters are needed for laser purification.Thus, to achieve a low fluorescence flux, spatial filtering processes(such as the oblique incidence method) can help improve fluorescencecollection efficiency and suppress background noise.

High speed and sensitivity Avalanche photodiodes (APD) are widely usedin low photon flux detection processes. In one exemplary embodiment ofthe present system, an APD (e.g., C5460-01 from Hamamatsu PhotonicsK.K.) serves as the fluorescence detector. As is seen in the exemplarydatasheet of FIG. 5, the amplification of the APD is approximately 0.15Gigavolt/W at 800 nm. Considering 520 nm as the central emissionwavelength for fluorescein, it can be estimated that the realamplification for fluorescence, according to the spectral responsecurve, is 0.05 Gigavolt/W (see FIGS. 6A and 6B).

Background noise of the APD is expected to be 6 pW according to thenoise equivalent power from the datasheet (FIG. 5). Therefore, thedetection limit of fluorescence is about 6 pW, without the aid ofelectronics (e.g., op-amp, frequency filters) or numerical signalenhancement approaches (e.g., signal processing such as FFT). Accordingto tests of the present system, the detection limit is equivalent to 0.3pm thickness of the protein layer conjugated with fluorescein, i.e. 0.3pg/mm² protein planar density on the disk.

Frequency response is another important parameter of the APD. Thisparameter sets the upper limit for the detection speed. As can be seenin FIG. 5, the signal frequency response is approximately 100 kHz, suchthat if the system scans 100 μm diameter protein spots on a biologicalcompact disk with 0.05 second spin period (20 Hz), the central frequencyfor the spot signal can be estimated to equal 60 kHz (i.e., 20 Hz×0.3m/0.1 mm) on the outer ring of disk. Therefore, a spin frequency of 20Hz would be acceptable for a scanning system operating with a C5460-01APD. If a higher spin frequency is demanded, then a lower amplificationbut higher response rate (such as C5460) could accommodate thisrequirement.

As mentioned above, the fluorescence detection limit can be extended ifthe APD's noise is carefully analyzed and exploited, particularly sincethe frequency band of the fluorescence signal can be separated from themajor band of the noise spectrum.

Noise in fluorescence systems tends to be dominated by amplifier noisebecause of the associated low photon flux and high gain of thesesystems. This noise can have a 1/f character at low frequencies, and mayhave white noise properties at higher frequencies. The change insignal-to-noise with a change in the detection bandwidth depends on thefrequency dependence. The different conditions are shown generically inFIG. 7.

In the exemplary embodiment of the current invention, the laser beampasses rapidly over a succession of protein spots. Therefore, thisembodiment constitutes a laser scanning configuration. Laser scanningcan be accomplished either by a linear raster of the laser beam whilethe target remains fixed, or the laser beam can remain fixed while thetarget moves. In our case, the laser remains fixed and the target moves.The rotation of the spinning disc brings the same protein spot back tothe probe laser many times. This represents high-speed sampling that hasa strong advantage in the signal-to-noise ratio for scanning systemsrelative to static measurement systems. To show the advantages ofhigh-speed spinning and scanning, we show how the signal-to-noise isimproved over static measurements for the case of 1/f noise.

Static Measurement with 1/f noise: For a static measurement, the signalfrom a target location is measured with an integration time T, afterwhich the laser is moved (or the target is moved) to a new location tobegin the next measurement. The effective sampling frequency in thiscase is f=1/T, and the effective bandwidth is BW=f=1/T. The noise in thesignal is given by:P _(N) =P _(f) *BW/f=P _(f)and the detection bandwidth cancels the 1/f component of the noise, andno advantage is obtained by averaging.

High-Speed Repetitive Measurements with 1/f noise: In this case, thesampling frequency is set by the transit time Δt from one spot on thedisc to the next so f=1/Δt. The detection bandwidth is set by theintegration time BW=1/T. The noise power is then given byP _(N) =P _(f) *BW/f=P _(f) *Δt/Twhich is made smaller by choosing a shorter transit time (higher speed)and integrating longer. The comparison of the spinning detection noiseto the static detection noise described above shows the clear advantagesof high-speed spinning that are embodied in the biological compact discconcept. These noise arguments hold equally for both fluorescence andinterferometry. The dual-mode detection we describe here thereforebenefits directly from the high-speed spinning in the presence of 1/fnoise.

The following discussion about the electrical field of the disk is basedon the condition that the incident light's polarization is parallel withthe disk's surface.

The surface of the biological compact disk is designed to enhancein-line and phase contrast sensitivities of the disk. This sensitivitycan be predicted by determining the reflection coefficient r of thesurface. Fluorescence sensitivity can also be determined by thereflection coefficient r. Because the analyte monolayer of the surfaceis thin (less than 10 nm, about 1/50 of the wavelength), the opticalproperties of the surface influence the fluorescence excitationefficiency, particularly since the surface electrical field isdetermined by the interference between the reflected light and theincident light. For example, when the reflection coefficient of themicroarray surface is −1, the surface will be at the standing wave'snode position. In this case, the electrical field is almost zero in theproximity of the surface so that the fluorophore will not be excited andthe biolayer will not contribute a phase change. On the other hand, whenthe reflection coefficient of the microarray surface is +1, the electricfield is a maximum in the proximity of the surface so that thefluorophore will be excited, and the biolayer contributes a maximumphase shift that would be detected in a phase-contrast detection system.

A primary concept for the current embodiment of the invention is theoptimum excitation of both fluorescence and phase-sensitive detection(either phase-contrast or in-line). In the case of r=+1, the maximumfield automatically gives the maximum fluorescence and maximum phasecontrast signal together. This is one exemplary embodiment of thecurrent invention.

In the case of in-line detection, there is a trade off between electricfield strength at the surface and the condition of phase quadrature thatmust be set by the substrate structure. For in-line interferometry, theoptimum phase condition is a pi/2 phase shift, but this phase conditionproduces half the electric field at the surface, which decreases boththe phase contribution of the biolayer for interferometric detection andthe fluorescence intensity. Therefore, a balance must be set in thedesign that keeps the surface field as large as possible, while alsokeeping a phase condition reasonably near to quadrature.

Because the fluorescence excitation efficiency is proportional to theintensity of the electrical field, if the incident light's amplitude isE, then the surface electrical field is(1+r)E cos ωton the disk surface, where r is the reflection coefficient. Thisrelationship helps to predict the fluorescence excitation efficiency dueto the optical property of the disk surface. As a result, the followingconclusions can be reached: (1) if r=−1, the excitation efficiency iszero; (2) if r=1, the excitation efficiency is maximized; (3) if r=0,the excitation efficiency is half of the maximum value; and (4) therequirement for r is quite loose. In most situations, fluorescenceexcitation efficiency is rather large. This provides freedom to design asuitable reflection coefficient to accommodate the interferometricchannel's sensitivity, since it is more rigorous for a suitable r.

The three-dimensional plots of FIG. 8 illustrate the relationshipbetween r and the channel's sensitivity. Since r is a complex quantity,it needs two dimensions (modulus and phase) to be expressed. FIG. 8Ashows the relationship between the fluorescence excitation efficiencyand the reflection coefficient of the biological compact disc. FIG. 8Bshows the relationship between the sensitivity of in-lineinterferometric channel and the reflection coefficient of the biologicalcompact disc. FIG. 8C shows the relationship between the sensitivity ofphase contrast interferometric channel and the reflection coefficient ofthe biological compact disc.

Biological compact disks used in this embodiment can include silicondisks coated with 100 nm of silica film. These disks are useful forcommercial applications, particularly because they are inexpensive tomanufacture yet still exhibit robustness and good sensitivity for allchannels. Such disks are particularly useful for in-line interferometricchannel sensitivity applications. With respect to fluorescence detectionapplications, since fluorescence excitation efficiency is ∝|1+r|², r canbe calculated by considering the following factors:

Incident angle 30 degrees Polarization direction parallel with disk'ssurface index of air 1 index of silicon dioxide 1.46313 index of Silicon4.379It can be calculated that r=0.27−0.24i. Therefore, |1+r|²=1.67. Thisvalue is quite large considering that the maximum value is 2. In thiscase, the fluorescence excitation coefficient should be good, while thephase shift is close to the pi/2 phase required for in-lineinterferometric detection.

The present embodiment has been tested with gel-printed protein gratingpatterns and spot-style immunoassays. The former pattern can provide aperiodic signal for system calibration and for the analysis of thesignal power spectrum. The latter shows the system's potentialapplications for biological research.

For the gel printed protein pattern, a physical adsorption method isused to immobilize protein molecules on the substrate surface. Accordingto this example, a hydrophobic activation was performed on the silicondioxide layer of the disk by surface silanization (the disks were soakedin 0.02M chlorooctadecylsilane Toluene solution for 12 hours). Theproteins adhere to the silanized disk surface through hydrophobicinteraction. Bovine serum albumin conjugated with fluorescein (A9771,Sigma corp.) is then printed on the disk in a grating pattern with a gelstamp method. The width of each protein stripe is about 100 um, and thegap between stripes is about 120 um. After printing, the surface of thedisk is rinsed with de-ionized water and then blown dry with purifiednitrogen to establish the protein layer as a monolayer. The results oftwo-channel scanning are shown in FIG. 9. On the same protein gratingpattern region (whose thickness is about 1˜4 nm—approximately amonolayer), an imaging scan is performed simultaneously with twochannels. FIG. 9A shows the light-scattering fluorescence, and FIG. 9Bshows the in-line interferometry illuminated at 488 nm. The crosscorrelation value between FIGS. 9A and 9B is 0.83, thereby showing thatthe two results are highly correlated in this case.

FIGS. 10A and 10B show the power spectra that correspond to FIGS. 9A and9B, respectively, using the Fast Fourier Transform Method. The spikes onthe spectrum shoulder come from the periodic protein stripe pattern. TheSignal-to-Background Ratio (SBR) can then be derived from the spectrumchart. Here, since it is known that the thickness of the protein layeris about 1-3 nm (monolayer), the SBR for the fluorescence andinterferometry channels are in the range of 300:1˜500:1. The detectionlimit for the lowest protein layer can be estimated as 1˜2.5 pm, i.e.1˜2.5 pg/mm² planar density, which is close to the detection limit(i.e., 0.3 pg/mm²), which is estimated from the APD detection limit asdiscussed above.

It was also found that the first ‘spike’, which is the fundamentalharmonic, is almost at the top of the spectrum shoulder, which exhibitsthe 1/f noise of the system (mostly originating from the APD). Thisindicates that target signal is not separated away from the 1/f noisefrequency domain on this sample. This is because of the motor's lowspinning frequency (20 Hz) and the relatively large distance between theprotein stripes. When scanning smaller samples (e.g., sub-millimeterspots with 80 Hz spinning frequency), the SBR could be improved by abouta factor of 10, which means that the detection limit can be extended to0.1˜0.25 pg/mm².

This embodiment's capacity to quantify immunoassays with high backgroundprotein concentration was then tested. Only the fluorescence andamplitude channels were used because they have the highest SBR. In thisexemplary illustration, the “sandwich model” immunoassay strategy isapplied to a biological compact disk (i.e., 100 nm silica coated silicondisk). To detect the target antigen's concentration in the solutionsample, which has a high background concentration, the correspondingantibody is immobilized on the disk, and then the disk is incubated withthe analyte solution. Consequently, the antibody binds with the targetantigen so that the antigen is anchored on the disk, while thebackground non-specific protein is washed off. When the target antigenis captured on the disk, the antigen can be incubated with afluorescein-conjugated antibody (for fluorescence detection) or anunconjugated antibody (for interferometric detection). The fluorescenceintensity or interferometric signal's increment is linearly related tothe antigen concentration in the original solution. Using a standardresponsive curve illustrating the relationship between the antigenconcentration and the signal increment, it is possible to acquire theantigen concentration quantitatively.

In an experimental procedure, eight wells of antibody spots are printedon an oxidized silicon disk. Each well includes a 2×2 array of spotsarranged in a unit-cell configuration. The unit-cell configuration forthis experiment comprises two spots on a first diagonal of anti-rabbitIgG, and two spots on the other diagonal of non-specific Horse IgG whichare a control. These eight wells are incubated respectively with 0,0.01, 0.03, 0.1, 0.3, 1, 3 and 10 ug/ml Rabbit IgG in 7 mg/ml rat lysateand then scanned. Thereafter, the spots are sequentially incubated with20 ug/ml anti-rabbit-biotin, 20 ug/ml avidin, 20 ug/ml anti-avidin, witha scan being performed after each incubation process.

The upper four rows of FIG. 11A show the thickness of the protein spotsacquired from the interferometric channel. The first row shows thethickness of the spots after incubation with the series of rabbit IgGsolutions in the concentration ladder. The second row shows thethickness of the spots after incubation with the anti-rabbit-biotin. Thethird row shows the thickness of the spots after incubation with theavidin. The fourth row shows the thickness of the spots after incubationwith the anti-avidin-FITC. The fifth row shows fluorescence signalsafter incubation with anti-avidin-FITC.

FIG. 11B shows the response curves for the analyte concentration ladderon both the fluorescence and interferometric channels. In FIG. 11B, thecurves show spot thickness increments after each incubation process as afunction of concentration. All of the curves are fitted with theLangmuir binding equation:

${\Delta\; d} = {C\frac{\lbrack{antibody}\rbrack}{K_{D} + \lbrack{antibody}\rbrack}}$where K_(D) is the dissociation constant between antigen and antibody,or between avidin and biotin-conjugated protein. In the three curves forthe interferometric channel, the increments increase monotonically withincreasing concentrations indicating that the detection limit is below10 ng/ml. The fluorescence response curve (upper solid curve) shows thesame trend. As such, this experiment shows that the system succeeds inreaching 0.01 ug/ml detection limits on both the fluorescence andinterferometric channels in the presence of 7 mg/ml complex proteinbackground.

Another exemplary embodiment comprises a four-channel detection methodfor protein-patterned biological compact disks that simultaneouslymeasures fluorescence, Rayleigh scattering and/or diffraction, and twointerferometric channels in orthogonal quadratures (i.e., a differentialphase channel and a direct phase channel). The latter two channelsconstitute label-free interferometric protein detection, whilefluorescence and Mie scattering detection provide complementary tools.

Optical biosensors normally include a probe light and one or moredetectors. When illuminated by probe light, protein molecules containinga fluorophore are excited and then emit fluorescence, or protein byitself scatters the probe light. By detecting fluorescence or scatteredlight, the protein information is obtained. For both cases, a discretedipole approximation can be used to analyze the absorption,fluorescence, or scattering due to molecules. One sub-wavelength sizemolecule is considered as one discrete dipole when fluorescence orscattering occurs. Subsequently, a protein agglomerate or a proteinlayer on a surface could be treated as a group of dipoles. Within thisapproximation, the optical properties of the four channels are analyzed.

Protein molecules are immobilized on the dielectric layers on thebiological compact disk with complex reflection coefficient r. In thesimplest model, molecules are distributed evenly (from a macroscopicview), and they are illuminated with a focused Gaussian laser beam whosewaist diameter is D. The polarization is parallel with the surface,shown as the arrow parallel to the x-axis in FIG. 12. Otherpolarizations are also possible. Every molecule is a discrete dipolewhen illuminated with the probe light.

FIG. 12A illustrates protein molecules on a biological compact diskbeing illuminated with a focused Gaussian beam. Polarization isindicated by the arrow in FIGS. 12A and 12B. Each molecule radiatesfluorescence or scatters probe light in the manner of one discreteelectric dipole. FIG. 12B illustrates a set of angular coordinates thatcan be used to calculate fluorescence, Rayleigh scattering (forinterferometric channels) and Mie scattering angular distribution ofintensity.

A protein molecule has an inherent dipole moment {right arrow over (P)}even before excitation. The excitation probability of this dipole isproportional to sin² θ cos² φ (where θ and φ are angles of {right arrowover (P)} in the angular coordinates, shown in FIG. 12B). Within themean lifetime (usually larger than 1 nanosecond) of the excited energylevel, the dipole emits one photon. The probability of emissiondirection is proportional to sin² α (where α is the angle between thedipole moment and emission direction). To simplify this model, it can beassumed that the dipole moments are oriented isotropically in space.Because the relaxation time of excited molecules is random, and has atleast one nanosecond variation, fluorescence from different molecules isincoherent. As a result, the fluorescence intensity distribution in thefar field equals the algebraic sum of all the dipole intensities in thefar field.

Under these conditions, the fluorescence intensity angular distributionin the far field is:

$\begin{matrix}{{F\left( {\theta,\phi} \right)} = \frac{d\;\sigma}{d\;\Omega}} \\{= {K{\int_{\phi = 0}^{2\pi}{\int_{\theta^{\prime} = 0}^{\pi}{\sin^{2}\theta^{\prime}\cos^{2}{\phi sin}^{2}\alpha{\mathbb{d}\Omega^{\prime}}}}}}} \\{= {K{\int_{\phi = 0}^{2\pi}{\int_{\theta^{\prime} = 0}^{\pi}{\sin^{2}\theta^{\prime}\cos^{2}{\phi\left( {1 - {\cos^{2}\alpha}} \right)}{\mathbb{d}\Omega^{\prime}}}}}}} \\{= {K{\int_{\phi = 0}^{2\pi}{\int_{\theta^{\prime} = 0}^{\pi}{\sin^{3}\theta^{\prime}\cos^{2}{\phi^{\prime}\left( {1 - \left( {{\sin\;{\theta cos\phi sin\theta}^{\prime}\cos\;\phi^{\prime}} +} \right.} \right.}}}}}} \\{\left. \left. {{\sin\;{\theta sin}\;{\phi sin\theta}^{\prime}\sin\;\phi^{\prime}} + {\cos\;{\theta cos}\;\theta^{\prime}}} \right)^{2} \right){\mathbb{d}\theta^{\prime}}{\mathbb{d}\phi^{\prime}}} \\{= {\frac{8\pi}{15}{K\left( {2 - {\sin^{2}{\theta cos}^{2}\phi}} \right)}}}\end{matrix}$where K is a constant. From this equation, it is obvious that thefluorescence intensity reaches a maximum when

${\phi = {\frac{\pi}{2}\mspace{14mu}{or}\mspace{14mu}\frac{3\pi}{2}}},$i.e. along the plane perpendicular to the polarization direction of theprobe light. This conclusion suggests the best fluorescence collectionposition. In the present system, the fluorescence collection lens isimmediately above an illuminated region while the probe light isincident obliquely at 30 degrees.

The reflection coefficient r of the biological compact disk surface alsoaffects the fluorescence sensitivity. Because the protein layer on thesurface is thin (less than 10 nm, about λ/50), the electromagneticboundary condition of the surface imposes a large influence onfluorescence excitation efficiency. This is because the surface electricfield is determined by interference between reflected and incidencelight. For example, when the reflection coefficient r=−1 on themicroarray surface, the surface will be at the nodal position of theresulting standing wave. In this case, the electric field is almost zeroin the proximity of the surface so that the fluorophore will not beexcited.

Fluorescence excitation efficiency is proportional to the magnitude ofthe electric field. If the incident light amplitude is E, then thesurface electric field is (1+r)E cos ωt on the disk surface, where thereflection coefficient r is a complex number. Therefore, thefluorescence excitation efficiency is proportional to |1+r|². Thefluorescence intensity angular distribution becomes:F(θ,φ)∝|1+r|²(2−sin² θ cos² φ)This equation is valid even after considering fluorescence reflected bythe dielectric surface.

Although both interferometric signals and fluorescence can be treated asdipole radiation from molecules, the optical properties have afundamental difference and thus have different intensity distributionswithin the solid angle. Interferometric signals arise from coherentRayleigh scattering. When illuminated with coherent probe light thedipole radiation superposes in the far field. The superposition causesthe scattered light to be strongest in the reflected (specular)direction in the far field. For a thin protein layer, the superposedfield calculated for dipole radiation coincides with the reflected lightcalculated using a thin film model. Therefore, to simplify computationthe protein layer is treated as a dielectric thin film.

Changes in the protein film changes the reflection coefficient of thebiological compact disk. Interferometric channels detect the presenceand thickness of the film by monitoring the reflection change. Thechange can be optimized by careful selection of r. The biologicalcompact disk surface coating is designed to optimize the interferometricand fluorescence channel sensitivities. To optimize the response, therelationship between the reflection coefficient r of the biologicalcompact disk and the reflection change due to a protein layer (see FIG.13A) is determined. FIG. 13A illustrates that the reflection change isproportional to the thickness of the protein layer when the proteinlayer on the surface of the disk is thin enough (much less than theprobe light wavelength). If the thickness of the protein layer is d, therefractive index is n_(p), and the reflection coefficient of thebiological compact disk surface is r, then the protein layer on thebiological compact disk has a new reflection coefficient r′ caused bythe protein layer which is solved with the matrix method for calculatingmultiple dielectric layers to be:

$r^{\prime} = \frac{{\left( {{\mathbb{e}}^{\mathbb{i}\delta} - {\mathbb{e}}^{- {\mathbb{i}\delta}}} \right)r_{0}} + {r\left( {{\mathbb{e}}^{\mathbb{i}\delta} - {r_{0}^{2}{\mathbb{e}}^{\mathbb{i}\delta}}} \right)}}{\left( {{\mathbb{e}}^{\mathbb{i}\delta} - {r_{0}^{2}{\mathbb{e}}^{- {\mathbb{i}\delta}}}} \right) + {{r\left( {{\mathbb{e}}^{- {\mathbb{i}\delta}} - {\mathbb{e}}^{\mathbb{i}\delta}} \right)}r_{0}}}$where r₀ is the reflection coefficient of the air-protein interface, and

$\delta = \frac{2\pi\; n_{p}d\;\cos\;\theta}{\lambda}$is the phase change caused by the protein layer (single pass). Usingthis relationship along with the original reflection coefficient r, thenew reflection coefficient r′, and the thickness of protein layer d, thepresence and mass areal density of the protein molecule can be detectedby monitoring the change of the reflection coefficient of the biologicalcompact disk. There are two interferometric channels to monitor thereflection change.

The amplitude channel directly detects the reflectance of the biologicalcompact disk. It is called “amplitude channel” because this channeldetects the intensity of the reflected radiation that interferes withthe light scattered by the protein molecules. Because of the conditionof phase quadrature that is established when the reflection coefficienthas a pi/2 phase shift, or nearly so, the phase associated with theprotein layer is transduced into intensity (amplitude) at the detector.When the system is scanning a protein layer, the reflectance change is:ΔI _(R) =I ₀(|r′| ² −|r| ²)If the thickness of the protein layer is thin (much less than the probelight wavelength), ΔI_(R) is approximately proportional to the proteinlayer thickness. With knowledge of r and the reflectance change, thethickness of the protein layer is calculated.

The phase-contrast channel detects the differential phase change of thereflection coefficient. When the system scans the edge of the proteinlayer, part of the focused spot is reflected with r while the other partis reflected with r′. In the far field, the reflected direction willslightly depart from the original direction, and the shifted angle isproportional to the phase difference between r and r′. A quadrantphotodetector (position-sensitive detector) is used to detect this angleshift. The detector sensing window is divided into four quadrants. Thecenter of the reflected light falls evenly on the center so that allquadrants have the same signal. When the reflection angle shifts, thephoton flux on the quadrants acquire a small difference. The relationbetween this difference and the thickness of the protein layer is:ΔI _(φ) =TI ₀(φ′−φ+2δ tan θ_(p)/tan θ₀ |r| ²where φ′ and φ are the phase of r′ and r, θ₀ is the incident angle,θ_(p) is the refraction angle in the protein layer, and T is thecoefficient which converts phase shift into center shift signal ΔI_(φ).Simulations calculate T to be approximately 0.5 in this embodiment.

FIG. 13B illustrates that if protein molecules agglomerate on thesurface, Mie scattering dominates, and the scattering can be detectablein the Mie scattering channel. In this embodiment, the Mie scatteringchannel shares the same optical path with the fluorescence channel, butit could have a separate lens and detector, or share the same lens anduse a beamsplitter to direct the scattering channel to a separatedetector. Usually, Rayleigh scattering is centered along the reflectiondirection because of the interference and the redistribution of thescattered electric field. But for larger agglomerations of proteinmolecules, if the agglomeration size is comparable or even larger thanthe wavelength of the probe light, then scattered light is detectableaway from the reflection direction (see FIG. 13B). This provides anopportunity to separate a scattered signal from light reflected by thedielectric surface to eliminate the background. With appropriatefilters, the system can switch between fluorescence and Mie scatteringchannels, or with the beamsplitter the two channels could be acquiredsimultaneously with separate detectors. The potential of the Miescattering channel can be further exploited because proteinagglomeration is a common phenomenon on a microarray surface. When theparticles are large, even Mie scattering can be predominantly in theforward direction. Therefore, in one embodiment, the Mie channelphotodetector could be situated on either side of the interferometricchannel. It is also possible to use different quadrants of a quadrantdetector to detect the Mie scattering and the interferometric channelsseparately. In this embodiment, the lower quadrants could be summed ordifferenced to obtain the in-line and phase-contrast signals,respectively, while the upper quadrant could be used to detect thelow-angle forward-scattered Mie scattering.

In the current embodiment, experiments were performed with a biologicalcompact disk having a multilayer dielectric stack structure of tenrepeated layers of SiO₂ and Ta₂0₅ with thicknesses of 113.4 nm and 72.2nm, respectively, on a glass substrate. Working under the condition of a30° obliquely incident s-polarized 488 nm laser beam, the surfacereflection coefficient is r=−0.58−0.35i. The fluorescence andinterferometric channels were appropriately optimized for thisbiological compact disk.

An oblique incidence design was established for this system to benefitfrom two distinct solid angular emission distributions due to thedifferent coherent properties of fluorescence and interferometricsignals. In oblique incidence, the probe laser beam is incidentobliquely on the biological compact disk, and fluorescence is collectedwith a convex lens above the disk. In this configuration, the reflectedlight does not enter the fluorescence collection lens, but fluorescencecan be acquired with high efficiency. Interferometric signals aredetected by acquiring the reflected probe light. In this way, two typesof signals are detected simultaneously without influencing each other.The reason for this design is that the fluorescence efficiency is verylow from the fluorophore-conjugated protein layer (1˜10 nm thickness) onthe surface. Empirically, the ratio of photon flux between fluorescenceand probe light is about 1:10⁷. If reflected probe light is mixed intothe fluorescence channel, the extremely strong background causes a largeinfluence on the fluorescence detection precision. Long-pass filtersalone may not be enough to eliminate the background. Spatial filtering,as from oblique incidence, improves the fluorescence collectionefficiency and suppresses background.

One embodiment of a four-channel microarray detection system is shownschematically in FIG. 14. This system is capable of simultaneouslyacquiring four different signals from protein molecules on a biologicalcompact disk. These four channels are: fluorescence and Mie scatteringchannel (detected by a high-amplification APD 90), amplitude and phasecontrast channel (interferometric channels, detected by a quadrantphotodiode 88). The embodiment illustrated in FIG. 14 comprises a laser80 (Innova300, Coherent Inc.), a linear stage 82 (MM2K, Newport), aspinning motor 84 (Scanner motor, Lincoln Laser, Inc.), a biologicalcompact disk 86, a quadrant detector 88 (PC50-6, Pacific Silicon SensorInc.), an APD 90 (C5460-01, Hamamatsu Company), an oscilloscope 92, acomputer 94 and optical components such as mirrors, filters and lenses.The system is now discussed with a focus on the following threecategories: light path, scanning mechanism, and electronics.

To map the entire disk, the scanning mechanism uses a polar coordinatesystem. The spin motor 84, on which the biological compact disk 86 ismounted, provides the angular coordinates when the motor 84 spins in aselectable frequency ranging from 20 Hz to 80 Hz. A linear stage 82provides the radial coordinate. In the experimental embodiment, thelinear stage 82 can move back and forth with 0.1 um linear precision and300 mm maximum travel distance. The spin motor 84 is fixed on the linearstage 82 so that two-dimensional mapping can be realized withappropriate control by the computer 94. This system is capable ofmapping a 100 mm diameter of the biological compact disk in 30 minuteswith 2 um by 2 um pixel resolution.

The illumination laser light emitted by the laser 80 has a wavelength of488 nm. The laser beam is steered and focused onto the surface of thebiological compact disk 86 with a filter, several mirrors and one 10 cmconvex lens. The radius of the focal spot is about 20 um on the disk 86.Higher resolution can be achieved by switching the 10 cm lens with ashort focal-length lens or a microscope objective lens. The reflectedlight is guided into the quadrant detector 88 which is responsible foracquiring the interferometric signals (amplitude and phase contrastchannels). A 4 cm convex lens above the biological compact disk 86gathers fluorescence or Mie scattering radiation and sends it to the APD90. A 510 nm long-pass optical filter 96 effectively blocks thescattered laser light for fluorescence detection. The long-pass opticalfilter 96 is removed from the optical path for detection of the Miescattering signal with this channel.

The oscilloscope 92 acquires waveforms for each scan track. The APD 90and the quadrant detector 88 are input into three channels of theoscilloscope 92 by coaxial cables. Two cables are connected to thequadrant detector 88 to acquire the two types of interferometric signals(i.e., amplitude and phase contrast). One cable is connected to the APD90 to sequentially acquire the fluorescence signal and the Miescattering signal depending on whether the long pass filter 96 is in theoptical path. One more coaxial cable connects the stage 82 to theoscilloscope 92 for the stage 82 to send a trigger signal to theoscilloscope 92. The computer 94 controls the linear stage 82 andrecords data from the channels of the oscilloscope 92.

This system has been tested with Gel-printed protein grating patternsand spotted patterns of antibodies. The former provide a periodic signalfor system calibration and signal power spectrum analysis. The lattershows the system detection for immunological assays.

In the gel-printed protein patterns, the protein molecules areimmobilized by physical adsorption following hydrophobic activation ofthe silicon dioxide surface of the disk by silanization (disks soak in0.02M chlorooctadecylsilane toluene solution for 12 hours). Proteinsbind with the silanized disk surface through hydrophobic interaction.Bovine serum albumin (BSA) conjugated with fluorescein (A9771, SigmaCorp.) is printed on the disk in a grating pattern with a gel stampmethod. Each protein stripe width is 100 um, and the gap between twostripes is 120 um. After printing, the disk surface is rinsed withdeionized water then blown dry with purified nitrogen. Because theprotein is conjugated with fluorescein (absorption wavelength of 492nm), the four-channel system is able to image the protein pattern inboth the fluorescence and the interferometric channels.

FIG. 15 shows the signals collected from the four channels with thisbiological compact disk. On the same region of the protein gratingpattern, whose thickness is about 1˜4 nm (approx. a monolayer), imagingis simultaneously performed with the four channels. FIG. 15A shows thefluorescence signal captured by the APD 90. FIG. 15B shows the Miescattering signal captured by the APD 90. FIG. 15C shows the amplitudesignal captured by the quadrant detector 88. FIG. 15D shows the phasecontrast signal captured by the quadrant detector 88.

The data in FIGS. 15A, 15C, and 15D show strong signals from thepatterned protein, while the Mie scattering data in FIG. 15B isvirtually blank. This means that protein molecules are printed evenlyand did not agglomerate. However in other experiments, strong Miescattering has been observed in this channel. Upon observing the Miescattering data more carefully, a “stain” was found near the top-rightcorner which could be due to a dust particle or agglomerated protein.The cross-correlation value is 0.83 between the fluorescence channel inFIG. 15A and the amplitude interferometry channel in FIG. 15C,demonstrating that the fluorescence and amplitude channels are highlycorrelated, although not identical, with the differences caused bydifferences between specific and nonspecific mass binding, and alsocaused by differences in fluorophore microenvironments on the disc.

FIGS. 16A-D show the power spectra corresponding to the data in FIGS.15A-D, respectively. The power spectra for fluorescence is shown in FIG.16A; the power spectrum for Mie scattering is shown in FIG. 16B; thepower spectrum for the amplitude interferometric channel is shown inFIG. 16C, and the power spectrum for the phase contrast interferometricchannel is shown in FIG. 16D. The family of peaks in the spectra comesfrom the periodic protein stripe pattern. The signal-to-background ratio(SBR) for the power spectra in FIGS. 16A, 16B, 16C and 16D arerespectively: 532:1, 1:1, 617:1, and 98:1. The amplitude channel and thefluorescence have similar SBR values, indicating that the sensitivitiesare almost equal in these cases. The phase-contrast channel SBR isrelatively low but still considered strong. The thickness of the proteinlayer is 1˜3 nm in this experiment. The detection limit for the lowestdetectable protein density is estimated to be 2˜6 pm, or about 2˜6pg/mm² areal density.

In the power spectra graphs of FIG. 16, the first-order signal frequencyis almost at the top of the spectrum shoulder, which arises from the 1/fnoise of the system combined with surface roughness of the disc. Thisindicates that the target signal may not be separated from the 1/f noisefrequency domain in this experiment. This is because of the low spinfrequency of the motor 84, about 20 Hz, and the relatively largedistance between the protein stripes. By scanning on smaller proteinpatterns, such as submilimeter spots, with 80 Hz spinning frequency theSBR can be improved by more than a factor of 10 which extends thedetection sensitivity to 0.2˜0.6 pg/mm².

It is important to note that 1/f noise is not equivalent to surfaceroughness. Noise is stochastic and changes from circuit to circuit ofthe disc. In contrast, surface roughness is a fixed property of the discand can be measured with the high accuracy of the interferometricmetrology. Therefore, this surface roughness is not noise, but can bemeasured accurately and subtracted accurately between a pre- and apost-scan that seeks to measure the amount of bound protein. It is whenthe surface is measured accurately and subtracted that the sensitivityof this technique achieves low values such as 0.2 to 0.6 pg/mm².

This embodiment of an integrated protein microarray detection system,can perform fluorescence, interferometry and Mie scatteringsimultaneously on a protein-patterned biological compact disk.Biological compact disk structures optimized for each channel werefabricated and tested with periodic protein patterns. The results showthat both interferometric and fluorescence channels can achieve a 5pg/mm² detection limit. The immunoassay experiment showed thefour-channel system potential for immunoassays with high-concentrationbackgrounds. The system detected 10 ng/ml target protein in 7 mg/mllysate.

In another embodiment we explore the difference between a forward and areverse assay. Fluorescence compared to interferometry shows importantdifferences in this comparison. This experiment is shown in FIG. 17. Theunit cells in this case have anti-goat antibody printed on one diagonalto bind antigen from sample, while the opposite diagonal has printedrabbit antigen to capture antibody from sample. The sample consists ofFITC-conjugated anti-rabbit cultured in goat. In a single incubationboth a forward and a reverse assay can be evaluated in both theinterferometric and the fluorescence channels on the two oppositediagonals. The incubations were made with increasing sampleconcentrations of 0, 0.03, 0.1, 0.3, 1, 3, 10, and 30 ug/ml. The spotintensities from the interferometry and fluorescence channels are shownin FIG. 17A. The response curves for both the forward and reverse assaysare shown in FIG. 17B. It is clear from the response curves that thereverse assay has a much stronger response than the forward assay. Thisgeneral trend is captured by both the interferometric and fluorescencechannels. However, there are quantitative differences betweeninterferometry and fluorescence that can highlight different mechanismsbetween forward and reverse assays.

To provide further calibration and correspondence between interferometryand fluorescence, a two-channel acquisition of backfilled proteinstripes at a concentration of 10 ug/ml is shown in FIG. 18. Thefluorescence channel is shown in FIG. 18A, and the interferometrychannel is shown in FIG. 18B, with the associated power spectra in FIG.18C. The correlation between the two channels is shown in FIG. 18D.There is clean separation between the bright and the dark stripes withstrong correlation in the corresponding values. These data show strongcross-validation of the interferometry and fluorescence channels.

To test the detection limits, stripes were backfilled at a concentrationof 10 ng/ml. The results are shown in FIG. 19. FIG. 19A shows thefluorescence channel, FIG. 19B shows the interferometric channel, FIG.19C shows the corresponding power spectra, and FIG. 19D shows thecorrelation. There is still separation between the positive and negativestripes in the correlation. This concentration is near the detectionlimit for this approach that uses gel printing. Inhomogeneities in thegel printing technique limit the sensitivity.

A key difference between interferometry and fluorescence is thequenching phenomenon that is associated with fluorescence but not withinterferometry. One of the drawbacks of fluorescence is the destructionof the fluorophore, called bleaching, during illumination. To illustratethe power of the present multi-mode detection system, the bleaching offluorescence was measured simultaneously in both an interferometric anda fluorescence channel. The results are shown in FIG. 20. FIG. 20A showsthe fluorescence channel and FIG. 20B shows the interferometry channel.In this experiment the radius of the probe beam was not changed. Thesame track was measured repeatedly. During the scan, the fluorophoreslowly quenched, seen in FIG. 20A with time increasing downward.However, in the interferometry channel seen in FIG. 20B there is nobleaching. This is seen in the graph in FIG. 20C. The interferometrychannel is flat with time, while the fluorescence is bleached. Thisserves to illustrate fundamental differences between interferometry andfluorescence that the current invention exploits.

An extensive demonstration of dual fluorescence and in-lineinterferometry is shown FIG. 21. In this example the disc was printedwith 25,000 protein spots. Half were anti-rabbit and the other half werecontrol spots. The disc was incubated with rabbit in a forward-phaseassay, then followed with the sandwich that had a fluorescent tag. Thisdemonstrates the differences between forward and sandwich assays in amicro-spot format. It also shows the use and comparison of thefluorescence channel to the interferometry channel. The second row ofFIG. 21 is the interferometry channel, and the bottom row is thefluorescence channel. The fluorescence only appears in the finalsandwich incubation.

On the disc, there were 3,400 antibody spots (anti-rabbit IgG, R2004,Sigma Company) and 3,400 control spots (anti-mouse IgG, R2004, SigmaCompany) printed on one region of the biological compact disc (one disccan hold 50,000 spots). Each antibody spot is adjacent to one controlspot. The spot diameter was 200 μm. FIG. 21 a shows one part of the6,800 spots. A two-channel scan (scan 1) was performed to record theinitial thickness and fluorescence of these spots (see FIGS. 21 b 1 andc 1, a small area of the microarray is shown for better viewing). Thebiological compact disc was incubated with 10 ng/ml rabbit IgG (I5006,Sigma Company) in PBST (PBS+0.05% Tween). Bovine serum at 100 μg/ml(B8655, Sigma Company) is spiked in the solution as background protein.A two-channel scan (scan 2) measured the interferometric and fluorescentsignal change due to the antigen binding (see FIGS. 21 b 2 and c 2). Asecondary antibody formed sandwich assay to evaluate the two-channeldetection limit. The biological compact disc is further incubated with 1μg/ml anti-rabbit-FITC (F9887, Sigma Company) in PBST. A third scan(scan 3) measured the two-channel response due to the secondary antibodybinding (see FIGS. 21 b 3 and c 3).

In the analysis, the interferometric channel tracks the specific bindingbetween anti-rabbit IgG and rabbit IgG. FIG. 22A shows the heightincrement of the antibody spots and control spots after incubation with10 ng/ml rabbit IgG solution. In a histogram of the height increments ofall spots, the centers of the specific and control Gaussiandistributions are separated by a difference of 0.097 nm. The standarddeviations of the two distributions are respectively 0.10 nm and 0.106nm. So the standard errors are 0.10/√{square root over (3400)}=0.0017 nmand 0.0018 nm. If the antibody spot thickness increment is linear toantigen level at low concentration, the detection limit ofinterferometric channel is estimated as 250 pg/ml for the forward-phaseassay. The fluorescence channel detects no signal at this stage becausethe antigen has no bound fluorophore.

FIG. 22B shows the height increment of the antibody and control spotscompared with the fluorescence signal of the antibody spots afterincubation with the anti-rabbit-FITC. The detection limit of theinterferometric channel is estimated as 71 pg/ml. The detection limit islower than forward-phase assay because one antigen can bind with severalantibodies in the sandwich assay. The detection limit of thefluorescence channel is estimated as 31 pg/ml.

From the statistical analysis in FIG. 23, the scaling capabilities ofboth the fluorescence and interferometry channels can be furtherdemonstrated by studying sub-populations of spots on the disc. Scalingis important for microarrays because it shows the potential forexpanding the numbers of assay on a disc for highly multiplexed assays.The change in the standard error as the population size changes providesinformation about spatial correlations on the disc. The scaling analysisis shown in FIG. 23. The scaling varies nearly as the square root of thenumber of elements in the populations. This indicates that spatialcorrelations are mostly absent on the disc. The similar scaling betweenthe fluorescence and interferometric channels provides importantcross-validation of these two channels.

While exemplary embodiments incorporating the principles of the presentinvention have been disclosed hereinabove, the present invention is notlimited to the disclosed embodiments. Instead, this application isintended to cover any variations, uses, or adaptations of the inventionusing its general principles. Further, this application is intended tocover such departures from the present disclosure as come within knownor customary practice in the art to which this invention pertains.

1. A multi-modal data acquisition system for detecting target materialon a biological reaction surface, the data acquisition systemcomprising: a radiation source for generating an incoming beam toilluminate the biological reaction surface, the incoming beam impingingon the biological reaction surface at an oblique incidence angle andproducing a reflected beam, the reflected beam being produced by theincoming beam reflecting off the biological reaction surface; aninterferometric detector for detecting an interferometric signal fromthe illuminated biological reaction surface, the reflected beam beingdirected to the interferometric detector, a fluorescence detector fordetecting a fluorescence signal from the illuminated biological reactionsurface; the fluorescence detector being positioned to substantiallyminimize the incidence of the reflected beam on the fluorescencedetector; and a processing system for receiving the interferometricsignal and the fluorescence signal and determining the presence orabsence of the target material on the biological reaction surface. 2.The multi-modal data acquisition system of claim 1, wherein thebiological reaction surface is a biological compact disk.
 3. Themulti-modal data acquisition system of claim 2, wherein the fluorescencedetector is positioned above the biological compact disk andsubstantially perpendicular to the plane of the biological compact disk.4. The multi-modal data acquisition system of claim 2, furthercomprising: a spin motor upon which the biological compact disk can bemounted, the spin motor being configured to rotate the biologicalcompact disk.
 5. The multi-modal data acquisition system of claim 4,further comprising: a linear stage for moving the biological compactdisk relative to the optical system comprising the illumination source,the interferometric detector and the fluorescence detector; wherein therotation of the biological compact disk by the spin motor and thetranslation of the biological compact disk relative to the opticalsystem by the linear stage creates a polar coordinate system that can beused for referencing any point on the biological compact disk.
 6. Themulti-modal data acquisition system of claim 1, wherein theinterferometric detector is a quadrant detector configured to acquireboth a phase contrast interferometric signal and an in-line quadratureinterferometric signal.
 7. The multi-modal data acquisition system ofclaim 1, wherein the processing system comprises: an oscilloscopeconnected to the interferometric detector for receiving theinterferometric signal and connected to the fluorescence detector forreceiving the fluorescence signal; and a computer connected to theoscilloscope for determining the presence or absence of the targetmaterial on the biological reaction surface.
 8. The multi-modal dataacquisition system of claim 1, further comprising: a long-pass opticalfilter removably positioned in front of the fluorescence detector toeffectively block the scattered illumination beam from impinging on thefluorescence detector when the optical filter is positioned in front ofthe fluorescence detector; and wherein the fluorescence detector iscapable of receiving both the fluorescence signal and a scatteringsignal, and the processing system is capable of receiving the scatteringsignal from the fluorescence detector; and wherein, when the opticalfilter is positioned in front of the fluorescence detector, thefluorescence detector receives the fluorescence signal, and when theoptical filter is removed from in front of the fluorescence detector,the fluorescence detector receives the scattering signal.